Telomere maintenance consists of a homeostatic system that helps protect the genome. The research in this proposal focuses on three interlinked aspects of this system in yeast: telomerase, the enzyme that replenishes telomeric DNA at chromosomal ends, the signaling that prompts and regulates its action at telomeres in cells, and telomere structure, which itself is also a major regulator of telomerase action. One goal of the planned research is to build up a self-regulating telomere from which the principles of telomere homeostasis can emerge. Aim #1 is to build the telomere in vitro from defined components, in order to understand the DNA-protein, and protein-protein, interactions necessary for telomere function. Defined complexes of telomeric proteins bound to telomeric DNA will be analyzed using biochemical, biophysical and structural methods, including electron microscopy. Among the interactions of telomerase is its dimerization, and this, and interactions of the core telomerase RNP with other factors, will be explored in Aim #2. which will test the roles of intermolecular regulatory interactions among telomerase components in the mechanism and control of telomerase action. The goal is to test models for higher-order complex functions of telomerase, and also to test the hypothesis that telomerase action in yeast is regulated through newly-identified phosphorylations of telomerase-associated proteins. Aim 3 will analyze the cause and mechanism of a newly-found early response-a G2/M cell-cycle delay-to deletion of telomerase, to dissect out whether the delay is induced by loss of telomerase and/or shorter telomeres, and to determine the genetic dependencies of this response. Aim 4 is to determine the roles of Tel2 and Tell (the yeast ATM ortholog) in telomere maintenance. We will test the role of Tel2 in bringing telomerase to telomeres, and explore new findings on the mechanism by which Tell promotes telomerase action through interactions with specific chromatin proteins. Significance: The mechanisms by which telomerase and telomeres carry out their functions are crucial for genomic stability and cellular health. This proposal uses the experimentally advantageous yeast system with the aim of further understanding these centrally important, conserved processes, which in humans have been implicated in effects on health and disease exerted at a fundamental cellular level.